Puhuang Flavonoids Having A Protective Effecton Vascular Endothelial Cells With Hypoxic Injury

Objective:As the first barrier of blood and tissue,vascular endothelial cells are located between the blood flow and vascular smooth muscle cells,which are ones of the cells firstly affected by anoxia,Puhuang flavonoids is one of the flavonoid compound that extracted from Puhuang. It has proved that Puhuang flavonoid having estrogen-like effects in the preliminary animal experiments. By now,a large number of studies have shown that estrogen can protect vascular endothelial cells. In order to further clarify the estrogen-like effect of Puhuang flavonoids, this study made a stable and reliable hypoxia model of vitro cell to simulate hypoxic micro-environment,based on successfully cultured human umbilical vein endothelial cells(HUVE-12). Explore the changes of cell number and contents of NO,ET and PGI2 to study the protective effect of Puhuang flavonoids for vascular endothelial cells under the anoxic conditions.Methods:Culture human umbilical vein endothelial cells (HUVE-12) in vitro,using homemade hypoxia device to induce injury of vascular endothelial cell. Culture cells in 25cm culture flask,and inoculate cells into 96-well plate by 2.5×104 per ml in the experiment. The cells were divided into:A control group:normal cultured cells group; B model group; a group simply cultured in anoxic condition; C anoxic cultured cells with Puhuang flavonoids 150ug/mL(high-dose group); D anoxic cultured cells with Puhuang flavonoids 100ug/mL(middle-dose group); E anoxic cultured cells with Puhuang flavonoids 50ug/mL(low-dose group); F anoxic cultured cells with 17-βestradiol 10-8moL/mL; 4 hours after cultured in the anoxic condition,observe the morphological changes of HUVE-12 cells under the microscope. Use MTT colorimetric method to observe the effect of Puhuang flavonoids on the activity of vascular endothelial cells. Collect cell supernatant and use enzyme-linked immunosorbent assay (ELISA) method to determine content of prostacyclin (PGI2), endothelin (ET-1) in the cell supernatant,and nitrate reductase method to test nitric oxide (NO) level in cell supernatant.Results:The hypoxia induced activity of HUVE-12 cells decreasing, morphological changes, NO,PGI2levels lowering in cell supernatant and ET-1 increasing。Compared with the normal control group,the difference was significant (P<0.01);MTT results showed that:Compared with anoxic group, Puhuang flavonoids high-dose group, low-dose group, positive control group has significant difference(P<0.01). Compared middle dose group with the anoxia group, the difference was significant (P<0.05); The difference within high, low and medium dose group was not significant (P>0.05); Compared with anoxia group,NO,ET-1 levels in Puhuang flavonoids high and medium dose group,positive control group had significant difference (P<0.01).Compared with the anoxia group,NO,ET-1 levels of Puhuang flavonoids low-dose group had significant difference(P<0.05).Compared with anoxia group,PGI2 content of Puhuang flavonoids high-dose group and positive control group had significant difference (P<0.01).Compared with anoxia group,PGI2 of Puhuang flavonoids medium-dose group had significant difference(P<0.05);Puhuang flavonoids can improve the activity of HUVE-12 cell under anoxia condition,reduce NO,PGI2 decreasing degree,inhibited the formation of ET-1.There was no significant difference between Puhuang flavonoids high-dose group and positive control group (P>0.05). The effect of increasing PGI2 in middle dose group was weaker than positive control group. The effect of increasing NO and inhibiting ET-1 formation was weaker than positive control group.Conclusion:1.Hypoxia has obvious damage to vascular endothelial cells. Puhuang flavonoids can improve the HUVE-12 cell activity under anoxic condition, increase NO, PGI2 levels in cell supernatant, lower levels of ET-1, which is similar to the protective effect of estrogen on vascular endothelial cells.2. The protective effect of Puhuang flavonoids on vascular endothelial cells depend on medicine density 3.And the previous animal studies of this topic has already proved Puhuang flavonoids to be an effective phytoestrogens with estrogen-like effect, so this experiment suggests Puhuang flavonoids having a protective effect on vascular endothelial cells with hypoxic injury.